Analysis of conserved residues of the human puromycin-sensitive aminopeptidase

The puromycin-sensitive aminopeptidase (ApPS) is a zinc metallopeptidase involved in the degradation of neuropeptides. Putative catalytic residues of the enzyme, Cys146, Glu338, and Lys396 were mutated, and the resultant mutant enzymes characterized. ApPS C146S exhibited normal catalytic activity, ApPS E338A exhibited decreased substrate binding, and ApPS K396I exhibited decreases in both substrate binding and catalysis. ApPS K396I and ApPS Y394F were analyzed with respect to transition state inhibitor binding. No effect was seen with the K396I mutation, but ApPS Y394F exhibited a 3.3-fold lower affinity for RB-3014, a transition state inhibitor, indicating that Tyr394 is involved in transition state stabilization.     

Neuron-specific aminopeptidase and puromycin-sensitive aminopeptidase in rat brain development

Neuron-specific aminopeptidase (NAP) and the ubiquitous puromycin-sensitive aminopeptidase (PSA) were compared in the rat hippocampus during early development. Hippocampus contains the highest amount of NAP determined by a fast-protein liquid chromatography-aminopeptidase analyzer using Leu -naphthylamide as substrate. Both enzymes were found in the hippocampus in all ages. NAP was lower in immature rat; the 19th embryonic-day fetus contained the least. It increased steeply during the prenatal through the early postnatal period, 9-fold by the first month. The rate of increase diminished subsequently, increasing 20% in the second month and 13% in the third. The age-dependent increase in NAP activity was parallel to its protein expression as determined by Western blot. The specific molecular activity (hydrolytic activity/NAP antigenicity) in newborn, 15-day-old, and 30-day-old rats were 1.00, 0.88, and 1.00, respectively. The PSA developmental profile without linear increase in activity was distinct from NAP. PSA activity was higher than NAP in decreasing order, 100–4 times, during the same development span. Similarly, different growth profiles for NAP and PSA were also found in the primary culture of developing cerebellar granule cells. Puromycin (1–5 M) blocked neurite outgrowth and caused apoptosis by nonantibiotic effects. Our data suggest that the synaptosome-enriched NAP plays a role in neuron growth, differentiation, and information programming.     

Cellular localization of puromycin-sensitive aminopeptidase isozymes

We developed monoclonal antibodies (mAbs) against two isozymes of a cytosolic puromycin-sensitive aminopeptidase (PSA-I and PSA-II) purified from chicken brain. The isozymes could be distinguished using Ouchterlony double-immunodiffusion and Western immunoblot. Their distribution in neuronal and glial cells as visualized by indirect immunofluorescence with these mAbs was found to differ: PSA-I was confined mostly to glial lysosomes; PSA-II showed fibrillar distribution in both types of nerve cells, but in disparate patterns. These results and our findings of peptide structural differences suggest that the two PSA isozymes are expressed differently in the nervous system.     

Fluorescent bioprobes for visualization of puromycin-sensitive aminopeptidase in living cells

Non-peptide, small-molecular, non-competitive inhibitors of puromycin-sensitive aminopeptidase (PSA), that is, 3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (PAQ-22, 3) and its 1N-methyl analogue (MPAQ-22 4), were structurally modified to afford fluorescent bioprobes, ANTAQ (5) and DAMPAQ-22 (6). The cellular localization of PSA could be visualized by the use of these fluorescent bioprobes.     

Cloning and sequence analysis of the aminopeptidase My gene from Mycoplasma salivarium

Abstract The complete nucleotide sequence of a major component of aminopeptidase My purified from Mycoplasma salivarium was determined. The protein gene encoded a protein consisting of 520 amino acids with a molecular mass of 58079 Da. The protein contained two tryptophan residues, one of which was encoded by UGA. A computer-aided homology search suggested that aminopeptidase My had properties similar to those of leucine aminopeptidase (EC 3.4.11.1).     

Cloning and nucleotide sequence of the Vibrio proteolyticus aminopeptidase gene.

The gene encoding the Vibrio proteolyticus aminopeptidase was cloned and sequenced and its amino acid sequence was deduced. The gene encodes a 54 kDa protein, larger than the previously reported size of 30 kDa for the purified aminopeptidase. Sequence alignments revealed a 43-45% homology with two other Vibrio sp. extracellular proteinases.     

A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.     

Studies on the subsite specificity of the rat brain puromycin-sensitive aminopeptidase

The specificity of the puromycin-sensitive aminopeptidase from rat brain was examined. Using L-alanyl-beta-naphthylamide as substrate Vmax of the reaction was shown to be pH independent over the range of 5.5-9.0, while Km exhibited a pKa of 7.7. This latter value corresponds to the pKa of the amino group of the substrate. Using X-Ala and X-Leu to examine the specificity of the P1 site it was found that Arg and Lys exhibit the highest affinity, followed by Met, Val, Leu, Trp, and Phe, which bind congruent to 5- to 20-fold less well. Although Km varied more than 20-fold within this series, Vmax showed considerably less variation. Significantly weaker binding was observed with a P1 Gly, Ala, Ser, or Pro with no binding detectable with a P1 Glu. The presence of a P'1 Leu compared to P'1 Ala results in an approximate 10-fold decrease in Km with little change in Vmax. The effect of varying P'1 residues was examined with the series Leu-X. In this case basic and hydrophobic amino acids, with the exception of Val, all exhibit nearly the same Km. The binding of Arg-Arg and Lys-Lys showed the same Km as obtained for Arg-Leu or Lys-Leu, respectively. When Leu-Ser-Phe was compared to Leu-Ser the P'2 residue led to a 100-fold decrease in Km and slightly less than a 5-fold increase in Vmax. In contrast the addition of a P'2 Met to Leu-Trp results in only a 3-fold decrease in Km and a 3-fold increase in Vmax. The results indicate a preference for a basic or hydrophobic residue in the P1 and P'1 sites and indicate subsite-subsite interactions which primarily affect binding.     

Degradation of tau protein by puromycin-sensitive aminopeptidase in vitro

Tau, a microtubule associated protein, aggregates into intracellular paired helical filaments (PHFs) by an unknown mechanism in Alzheimer's disease (AD) and other tauopathies. A contributing factor may be a failure to metabolize free cytosolic tau within the neuron. The buildup of tau may then drive the aggregation process through mass action. Therefore, proteases that normally degrade tau are of great interest. A recent genetic screen identified puromycin-sensitive aminopeptidase (PSA) as a potent modifier of tau-induced pathology and suggested PSA as a possible tau-degrading enzyme. Here we have extended these observations using human recombinant PSA purified from Escherichia coli. The enzymatic activity and characteristics of the purified PSA were verified using chromogenic substrates, metal ions, and several specific and nonspecific protease inhibitors, including puromycin. PSA was shown to digest recombinant human full-length tau in vitro, and this activity was hindered by puromycin. The mechanism of amino terminal degradation of tau was confirmed using a novel N-terminal cleavage-specific tau antibody (Tau-C6g, specific for cleavage between residues 13-14) and a C-terminal cleavage-specific tau antibody (Tau-C3). Additionally, PSA was able to digest soluble tau purified from normal human brain to a greater extent than either soluble or PHF tau purified from AD brain, indicating that post-translational modifications and/or polymerization of tau may affect its digestion by PSA. These results are consistent with observations that PSA modulates tau levels in vivo and suggest that this enzyme may be involved in tau degradation in human brain.     

Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain

The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.     

Genetic analysis of dPsa, the Drosophila orthologue of puromycin-sensitive aminopeptidase, suggests redundancy of aminopeptidases

Abstract. The Drosophila genome contains a single orthologue of mammalian puromycin-sensitive aminopeptidases, dPsa. Even though dPsa was expressed in many tissues during development, animals lacking dPsa activity were viable. Ubiquitous overexpression of dPsa during embryonic or larval development resulted in lethality and overexpression in isolated tissues during development resulted in localized lesions. These results suggest that even though dPsa function was not essential for viability, dPsa expression must be tightly regulated for normal development. By screening the Drosophila genome we found 43 predicted aminopeptidases and generated a phylogenetic tree of aminopeptidases related to dPsa by sequence. We discuss possible functions of dPsa and the idea that other Drosophila aminopeptidases might perform redundant functions with dPsa for regulating protein turnover.     

Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC

Abstract A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-β-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50 909 Da. The enzyme is functional and extremely overexpressed in E. coli .     

Identification of a puromycin-sensitive aminopeptidase from zebrafish (Danio rerio)

An aminopeptidase from zebrafish (Danio rerio) was purified 1247-fold to homogeneity with 35.4% recovery by column chromatography successively on DEAE-sephacel, hydroxyapatite, and phenyl-sepharose. The molecular mass of the enzyme was estimated at 98 kDa by SDS-PAGE and gel filtration. Optimum temperature and pH of the enzyme were 45°C and 7.5, respectively. The enzyme preferentially hydrolyzed substrate Leu-MCA with k(cat)/K(m) of 4.2×10(6)M(-1)s(-1) and an activation energy of 68.9 kJ M(-1) [corrected], respectively. It was specifically inhibited by bestatin, puromycin and metal-chelating agents, and Zn(2+) seemed to be its metal cofactor(s). Some l-amino acids significantly inhibited its activity, and l-cysteine was a non-competitive inhibitor with a K(i) of 0.27 mM. According to the peptide mass fingerprint analysis, the enzyme was highly matched with the predicted D. rerio aminopeptidase puromycin sensitive (gi: 255683530) (EC 3.4.11.14), suggesting that the present enzyme is a puromycin-sensitive aminopeptidase of zebrafish.     

Purification and characterization of a solvent stable aminopeptidase from Pseudomonas aeruginosa: Cloning and analysis of aminopeptidase gene conferring solvent stability

Aminopeptidase from a solvent tolerant strain Pseudomonas aeruginosa PseA was purified and studied for its biochemical and molecular characteristics. Ion-exchange chromatography resulted in 11.9-fold purification and 38% recovery of the 56 kDa enzyme. The enzyme was found to be stable over a pH range of 6.0–8.0 and appreciably thermostable up to 70 °C. PseA aminopeptidase exhibited K_m of 3.02 mM and V_max of 6.71 μmol/mg/min towards l-Leu-p-nitroanilide. Remarkable stability in both hydrophilic and hydrophobic solvents makes PseA aminopeptidase unique. Partial N-terminal sequence of enzyme showed exact match with probable aminopeptidase of P. aeruginosa PAO1, coded by gene pepB. Polymerase chain reaction amplified the 1611-bp open reading frame encoding a 57.51 kDa, 536 amino acid PseA PepB polypeptide. The deduced PseA PepB protein sequence contained a 24-residue signal peptide (2.57 kDa) followed by a 1.28 kDa propeptide and a mature product of 500 residues. Search for conserved domain in PseA aminopeptidase explored its place in zinc-metallopeptidase family. Primary sequence analysis showed the hydrophobic inclination of the protein; and the 3D structure modeling elucidated the presence of a high content of hydrophobic residues on its surface probably imparting solvent stability to it. The enzyme might find potential applications in non-aqueous enzymology due to its marked thermostability and striking solvent stability.     

Cloning and nucleotide sequence analysis of the human beta-microseminoprotein gene

Beta-microseminoprotein (MSP) is a small protein (94 amino acids) synthesized by the epithelial cells of the prostate gland and secreted into the seminal plasma. Restriction endonuclease mapping of human genomic DNA with a human MSP cDNA probe identified a 19 kilobase (Kb) hybridizing band in both EcoRI and BamHI digestions. Subsequently, the 19 Kb EcoRI fragment of human genomic DNA containing the MSP gene was isolated and cloned into an EMBL4 phage vector. Screening of the recombinant phages resulted in the isolation of one clone containing the MSP gene. Restriction endonuclease mapping and sequence analysis of this clone revealed the human MSP gene of approximately 15 Kb in length. The gene contains four exons and three large introns of approximately 6, 1, and 7 Kb.     

Cloning and DNA sequence analysis of an X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. lactis NCDO 763.

Lactococcus lactis subsp. lactis NCDO 763 (also designated ML3) possesses an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). X-PDAP mutants were selected by an enzymatic plate assay on the basis of their inability to hydrolyze an L-phenylalanyl-L-proline-beta-naphthylamide substrate. A DNA bank from L. lactis subsp. lactis NCDO 763 was constructed in one of these X-PDAP mutants, and one clone in which the original X-PDAP phenotype was restored was detected by the enzymatic plate assay. The X-PDAP gene, designated pepXP, was further subcloned and sequenced. It codes for a protein containing 763 residues. Comparison of the amino-terminal sequence of the X-PDAP enzyme with the amino acid sequence deduced from the pepXP gene indicated that the enzyme is not subjected to posttranslational modification or exported via processing of a signal peptide. The pepXP gene from L. lactis subsp. lactis NCDO 763 in more than 99% homologous to the pepXP gene from L. lactis subsp. cremoris P8-2-47 described elsewhere (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and is also conserved in other lactococcal strains.     

Cloning and DNA sequence analysis of an X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. lactis NCDO 763.

Lactococcus lactis subsp. lactis NCDO 763 (also designated ML3) possesses an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). X-PDAP mutants were selected by an enzymatic plate assay on the basis of their inability to hydrolyze an L-phenylalanyl-L-proline-beta-naphthylamide substrate. A DNA bank from L. lactis subsp. lactis NCDO 763 was constructed in one of these X-PDAP mutants, and one clone in which the original X-PDAP phenotype was restored was detected by the enzymatic plate assay. The X-PDAP gene, designated pepXP, was further subcloned and sequenced. It codes for a protein containing 763 residues. Comparison of the amino-terminal sequence of the X-PDAP enzyme with the amino acid sequence deduced from the pepXP gene indicated that the enzyme is not subjected to posttranslational modification or exported via processing of a signal peptide. The pepXP gene from L. lactis subsp. lactis NCDO 763 in more than 99% homologous to the pepXP gene from L. lactis subsp. cremoris P8-2-47 described elsewhere (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and is also conserved in other lactococcal strains.